The poxviruses comprise a major virus family of medical, ecological and agricultural importance. The most notorious family member, smallpox has been one of the great killers of mankind. Although the disease was eradicated some 40 years ago, the possibility of smallpox re- appearance at some future time has increased immeasurably with the recent demonstration that a poxvirus very similar to smallpox could be recreated de novo, in the laboratory, with ease. Moreover, eradication and the cessation of vaccination has coincided with the appearance of feral human poxviruses including human monkeypox in Africa, the US and UK. Not knowing the lethal factor in smallpox, the full potential of such outbreaks remains uncertain. The importance of virus envelope and capsid proteins in mediating the effects of antiviral therapeutics and vaccines is undisputed. For small RNA viruses in particular, an understanding of virion structure at molecular or atomic resolution has instructed the development of therapeutic agents and an understanding of mechanisms of infection and disease. Due to their complexity, asymmetry and heterogeneity, poxvirus virions have, however, persistently eluded attempts to elucidate their molecular structure, closing a potential avenue of rational design and intervention. The P.I. hypothesizes that the relative complexity of the vaccinia virion may be a therapeutic Achilles heel. Moreover, a molecular-level understanding of virion morphogenesis and organization, one of the last remaining black boxes in the lifecycle of the poxviruses, impinges upon at least five of the seven classical stages of virus replication. A major gap in our knowledge of pox virion structure lies at the level of molecular architecture ? an intervening organizational level between ultrastructural features and the inventory of protein molecules contained within the virion. The P.I. has successfully applied a protein-protein chemical crosslinking approach in combination with protein mass spectrometry (XLMS) to discover neighboring proteins and domains within the undisrupted vaccinia virion in situ. Aim 1 of this proposal seeks to deepen the XLMS dataset to a level that will allow protein molecular docking. Combining XLMS with mutant virus particles blocked in morphogenesis and displaying no apparent internal organization, Aim 2 of this proposal asks whether the virion morphogenic pathway follows a classical programmed linear hierarchy or a process of self-organization with no single, dominant route from molecular components to assembled virion. Using ?QconCAT? quantitative MS, Aim 3 seeks to convert XLMS data to a molecular model by determining the global stoichiometries of virion proteins.